Role of peptidoglycan-active enzymes in the assembly of bacterial motility, secretion and efflux systems

肽聚糖(PG)是细菌细胞壁的主要结构成分，但在高等生物中不存在。它是一种坚固的链条栅栏状材料，孔径约100 kDa。它完全包围细菌，提供对高内部压力的保护，并有助于形成独特的细菌细胞形状。虽然PG对细菌的生存是必不可少的，几十年前就被确定为最早的抗生素的靶标，但对其合成的分子细节只有部分了解。在一定程度上，这种缺乏理解的原因是，大多数细菌都有多种非必要的PG活性酶，这些酶的活性似乎是多余的，这使得为单个蛋白质分配特定功能具有挑战性。青霉素等抑制PG合成的药物往往同时针对多个酶，因此很难确定是否一种或多种蛋白质的丢失是导致观察到的杀伤效果的原因。在物理方面，PG是一种单一的、巨大的共价连接聚合物，很难在了解单个蛋白质的贡献所需的分辨率水平上进行表征。我们需要知道的事情包括单个蛋白质做什么，它们何时何地做，以及它们的活动如何影响通过PG层到达细胞外部的大型蛋白质复合体的组装。这类复合体包括那些参与运动、蛋白质分泌和有毒化合物外流等关键过程的复合体。利用缺乏单一PG活性酶的细菌，我们进行了一项长期的研究计划，以了解PG代谢和结构的控制如何与跨越细胞壁的蛋白质复合体的组装相交。通过表型筛选，我们已经确定了动力和分泌系统正常组装所需的酰胺酶和PBP，以及作为细胞壁损伤哨兵并在药物外排泵组装中发挥作用的不同PBP和裂解转糖基酶，这两个过程都会导致抗生素耐药性。我们的研究将带来克服抗生素耐药性的新策略，这是一个日益严重的威胁加拿大人健康的问题，因为加拿大已经出现了对所有已知抗生素具有耐药性的菌株。

Peptidoglycan (PG) is a major structural component of bacterial cell walls but is absent in higher organisms. It is a strong, chain link fence-like material with a pore size of ~100 kDa. It completely surrounds bacteria, providing protection against high internal pressures and contributing to the formation of distinct bacterial cell shapes. Although PG is essential for bacterial viability and was identified decades ago as the target of the earliest antibiotics, the molecular details of its synthesis are only partly understood. In part, this lack of understanding has arisen because most bacteria have multiple, non-essential PG active enzymes with seemingly redundant activities, making it challenging to assign specific functions to individual proteins. Drugs such as penicillin that inhibit PG synthesis often target multiple enzymes simultaneously, making it hard to determine if loss of one or more proteins is responsible for the observed killing effects. On the physical side, PG is a single, gigantic covalently linked polymer that has been difficult to characterize at the level of resolution needed to understand the contribution of individual proteins. Among the things we need to know are what individual proteins do, when and where they do it, and how their activities affect the assembly of large protein complexes that pass through the PG layer to the outside of the cell. Such complexes include those involved in the key processes of motility, protein secretion and efflux of toxic compounds. Using bacteria lacking single PG-active enzymes, we have undertaken a long term research program to understand how the control of PG metabolism and structure intersects with assembly of cell-wall-spanning protein complexes. Through phenotypic screens, we have identified an amidase and a PBP that are required for normal assembly of motility and secretion systems; and a different PBP and a lytic transglycosylase that act as sentinels of cell wall damage as well as having roles in the assembly of drug efflux pumps, both processes that lead to antibiotic resistance. Our research will lead to new strategies to overcome antibiotic resistance, an increasingly serious problem threatening the health of Canadians, as strains resistant to all known antibiotics are already here.